Hepatocyte growth factor (HGF) is a broad spectrum mitogen for a variety of cells and is ubiquitously expressed by stromal fibroblasts. Structurally, it resembles plasminogen and consists of four N-terminal kringle domains and a C-terminal protease-like domain. HGF is synthesized as an 87-kDa protein and can be cleaved into a heterodimeric form consisting of a heavy and light chain held together by disulphide bonds. To provide working quantities of this growth factor for our ongoing studies, we expressed HGF using the baculovirus system with an average yield of approximately 0.5 mg per liter of conditioned medium. Recombinant HGF retained all the biological activities of the naturally purified growth factor. Northern analysis revealed at least four HGF-related transcripts (6.0, 3.0, 2.2, 1.3 kb) in human fibroblast cell line M426. cDNA cloning and sequencing showed at least three classes of HGF cDNA clones that were generated by an alternative splicing mechanism. They represented HGF molecules with sequences of (1) the full coding region. (2) the first two kringle domains (HGF/NK2). and (3) the first kringle domain (HGF/NK1). We purified to homogeneity the HGF/NK2 protein and demonstrated that it is nonmitogenic but that it could act as an HGF antagonist by competitive binding to the HGF receptor. Its role in HGF-mediated processes will be the focus for future studies.